Total Internal Reflection Fluorescence (TIRF) illumination and photokinetics (PK) illumination are two laser-based modes of illumination that are commonly used in microscopy. A typical TIRF system involves a focused laser beam near the extreme edge of the back focal plane (BFP) of a high numerical aperture (NA) objective lens. This arrangement results in a collimated beam of light that impinges upon the coverslip/sample interface at a steep angle of incidence. If the angle of incidence is steep enough, the light will be totally internally reflected at the coverslip (glass) to sample (water) interface. In this way, only the fluorescent particles that are located proximal (<100 nm) to the coverslip/sample interface are illuminated, thus greatly improving image contrast. Scanning TIRF is an advanced form of TIRF illumination in which the beam is scanned in a circular pattern around the edge of the objective BFP. This technique provides the thin illumination section of conventional TIRF, but greatly reduces many of the artifacts that plague TIRF microscopy including shadowing, scattering, and interference patterns. Additionally, scanning the excitation beam around the back focal plane reduces or eliminates these same artifacts in a conventional laser epifluorescent (non-TIRF) illumination scheme.
In contrast, PK illumination involves a collimated beam of light entering the objective lens so that the beam is brought to a tight focus at the sample plane. This focused beam can be used to cause a number of reactions at precise locations in the sample including but not limited to photoactivation, photobleaching, chemical uncaging, and laser ablation. Scanning PK provides the ability to steer the focused beam to any location within the sample plane without moving the sample itself.
It is desirable to provide both scanning TIRF and PK modes of illumination on a single instrument from a single laser launch point. Furthermore, many experiments can benefit from the ability to rapidly switch between these two modes.